Our newsletter informs about the latest news in quantitative real-time
PCR (qPCR and qRT-PCR), which are compiled and summarised on the Gene
Quantification homepage. The focus of this newsletter issue is:

BACKGROUND: Currently, a lack of consensus exists on how best to
perform and interpret quantitative real-time PCR (qPCR) experiments.
The problem is exacerbated by a lack of sufficient experimental detail
in many publications, which impedes a reader's ability to evaluate
critically the quality of the results presented or to repeat the
experiments.

CONTENT: The Minimum Information for Publication of Quantitative Real-
Time PCR Experiments (MIQE) guidelines target the reliability of
results to help ensure the integrity of the scientific literature,
promote consistency between laboratories, and increase experimental
transparency. MIQE is a set of guidelines that describe the minimum
information necessary for evaluating qPCR experiments. Included is a
checklist to accompany the initial submission of a manuscript to the
publisher. By providing all relevant experimental conditions and assay
characteristics, reviewers can assess the validity of the protocols
used. Full disclosure of all reagents, sequences, and analysis methods
is necessary to enable other investigators to reproduce results. MIQE
details should be published either in abbreviated form or as an online
supplement.

SUMMARY: Following these guidelines will encourage better
experimental practice, allowing more reliable and unequivocal
interpretation of qPCR results.

As part of the MIQE guidelines - chapter 5.1 - the RNA quality control
is an essential step in the quantification process.

5. Nucleic acid quality control
5.1. RNA samples
Since it is advisable to use approximately the same amount of RNA for
cDNA synthesis when comparing different samples, quantification of RNA
in extracted samples is important. However, there are several
procedures in common use, including spectrophotometry (Nanodrop),
microfluidic analysis (Agilent BioAnalyser, BioRad Experion),
capillary gel electrophoresis (Qiagen QIAexcel) or fluorescent dye
detection (Ribogreen); all produce different results making it unwise
to compare data obtained using the different methods. The preferred
method for RNA quantity determination uses fluorescent RNA binding
dyes, for example RiboGreen, which are best for the detection of low
target concentrations. In any case, it is advisable to measure all
samples using one method only and to report this
information. ... ... ...

PowerNest is a software tool enabling experimenters to explore the
effect of sampling on noise propagation throughout qPCR assays. The
sampling process is assumed to be comprised of a number of levels; the
acquisition of a sample and the preparation of extracted material,
reverse-transcription of the mRNA, and the qPCR itself. Given a small
set of data, representative of a larger assay, the error at each stage
of the experiment is profiled using a nested-ANOVA.
Armed with this information, PowerNest allows the experimenter to
explore the effects of modifications to the experimental design on the
expected total error of the assay. When given the financial cost of
replicates at each level, PowerNest will calculate a cost-optimal
sampling-plan, delivering an experiment design that will minimise
processing error and maximise the statistical resolution of the assay.
The software is temporarily undergoing final testing, during which
time it has been made available as a free download =>[Only registered users see links. ]

At the TATAA Biocenter Germany we offer qPCR application workshops, a
3-day qPCR Core Module and a 2-day qPCR Biostatistics Module. All
courses are held regularly in Göteborg, Sweden, in English and in
Freising-Weihenstephan, Germany, in German and English, and in Prague,
Czech Republic in English and Czech.
Depending on the occasion the workshop language and the different
prices may apply. Further customized workshops and specialized
trainings will be held as well across Europe and world-wide.
TATAA Biocenter Germany workshops are held in cooperation with BioEPS
GmbH, located at the campus of the Technical University of Munich, in
Freising-Weihenstephan, very close to the Munich Airport (MUC). For
more information and registration, please see our web page:
=> [Only registered users see links. ]